Cargando…
The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa
Human DNA polymerase kappa (hpol κ) is the only Y-family member to preferentially insert dAMP opposite 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) during translesion DNA synthesis. We have studied the mechanism of action by which hpol κ activity is modulated by the Werner syndrome protein (WRN),...
Autores principales: | , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2014
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231769/ https://www.ncbi.nlm.nih.gov/pubmed/25294835 http://dx.doi.org/10.1093/nar/gku913 |
_version_ | 1782344484926783488 |
---|---|
author | Maddukuri, Leena Ketkar, Amit Eddy, Sarah Zafar, Maroof K. Eoff, Robert L. |
author_facet | Maddukuri, Leena Ketkar, Amit Eddy, Sarah Zafar, Maroof K. Eoff, Robert L. |
author_sort | Maddukuri, Leena |
collection | PubMed |
description | Human DNA polymerase kappa (hpol κ) is the only Y-family member to preferentially insert dAMP opposite 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) during translesion DNA synthesis. We have studied the mechanism of action by which hpol κ activity is modulated by the Werner syndrome protein (WRN), a RecQ helicase known to influence repair of 8-oxo-dG. Here we show that WRN stimulates the 8-oxo-dG bypass activity of hpol κ in vitro by enhancing the correct base insertion opposite the lesion, as well as extension from dC:8-oxo-dG base pairs. Steady-state kinetic analysis reveals that WRN improves hpol κ-catalyzed dCMP insertion opposite 8-oxo-dG ∼10-fold and extension from dC:8-oxo-dG by 2.4-fold. Stimulation is primarily due to an increase in the rate constant for polymerization (k(pol)), as assessed by pre-steady-state kinetics, and it requires the RecQ C-terminal (RQC) domain. In support of the functional data, recombinant WRN and hpol κ were found to physically interact through the exo and RQC domains of WRN, and co-localization of WRN and hpol κ was observed in human cells treated with hydrogen peroxide. Thus, WRN limits the error-prone bypass of 8-oxo-dG by hpol κ, which could influence the sensitivity to oxidative damage that has previously been observed for Werner's syndrome cells. |
format | Online Article Text |
id | pubmed-4231769 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-42317692014-11-21 The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa Maddukuri, Leena Ketkar, Amit Eddy, Sarah Zafar, Maroof K. Eoff, Robert L. Nucleic Acids Res Genome Integrity, Repair and Replication Human DNA polymerase kappa (hpol κ) is the only Y-family member to preferentially insert dAMP opposite 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) during translesion DNA synthesis. We have studied the mechanism of action by which hpol κ activity is modulated by the Werner syndrome protein (WRN), a RecQ helicase known to influence repair of 8-oxo-dG. Here we show that WRN stimulates the 8-oxo-dG bypass activity of hpol κ in vitro by enhancing the correct base insertion opposite the lesion, as well as extension from dC:8-oxo-dG base pairs. Steady-state kinetic analysis reveals that WRN improves hpol κ-catalyzed dCMP insertion opposite 8-oxo-dG ∼10-fold and extension from dC:8-oxo-dG by 2.4-fold. Stimulation is primarily due to an increase in the rate constant for polymerization (k(pol)), as assessed by pre-steady-state kinetics, and it requires the RecQ C-terminal (RQC) domain. In support of the functional data, recombinant WRN and hpol κ were found to physically interact through the exo and RQC domains of WRN, and co-localization of WRN and hpol κ was observed in human cells treated with hydrogen peroxide. Thus, WRN limits the error-prone bypass of 8-oxo-dG by hpol κ, which could influence the sensitivity to oxidative damage that has previously been observed for Werner's syndrome cells. Oxford University Press 2014-10-29 2014-10-07 /pmc/articles/PMC4231769/ /pubmed/25294835 http://dx.doi.org/10.1093/nar/gku913 Text en © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Genome Integrity, Repair and Replication Maddukuri, Leena Ketkar, Amit Eddy, Sarah Zafar, Maroof K. Eoff, Robert L. The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa |
title | The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa |
title_full | The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa |
title_fullStr | The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa |
title_full_unstemmed | The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa |
title_short | The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa |
title_sort | werner syndrome protein limits the error-prone 8-oxo-dg lesion bypass activity of human dna polymerase kappa |
topic | Genome Integrity, Repair and Replication |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231769/ https://www.ncbi.nlm.nih.gov/pubmed/25294835 http://dx.doi.org/10.1093/nar/gku913 |
work_keys_str_mv | AT maddukurileena thewernersyndromeproteinlimitstheerrorprone8oxodglesionbypassactivityofhumandnapolymerasekappa AT ketkaramit thewernersyndromeproteinlimitstheerrorprone8oxodglesionbypassactivityofhumandnapolymerasekappa AT eddysarah thewernersyndromeproteinlimitstheerrorprone8oxodglesionbypassactivityofhumandnapolymerasekappa AT zafarmaroofk thewernersyndromeproteinlimitstheerrorprone8oxodglesionbypassactivityofhumandnapolymerasekappa AT eoffrobertl thewernersyndromeproteinlimitstheerrorprone8oxodglesionbypassactivityofhumandnapolymerasekappa AT maddukurileena wernersyndromeproteinlimitstheerrorprone8oxodglesionbypassactivityofhumandnapolymerasekappa AT ketkaramit wernersyndromeproteinlimitstheerrorprone8oxodglesionbypassactivityofhumandnapolymerasekappa AT eddysarah wernersyndromeproteinlimitstheerrorprone8oxodglesionbypassactivityofhumandnapolymerasekappa AT zafarmaroofk wernersyndromeproteinlimitstheerrorprone8oxodglesionbypassactivityofhumandnapolymerasekappa AT eoffrobertl wernersyndromeproteinlimitstheerrorprone8oxodglesionbypassactivityofhumandnapolymerasekappa |